The invention relates to pokeweed antiviral protein and expression of nucleic acids encoding various PAP mutants in transgenic plants. Many commercially valuable agricultural crops are prone to infection by plant viruses. These viruses are capable of inflicting significant damage to a crop in a given season, and thus can drastically reduce its economic value. The reduction in economic value to the farmer in turn results in a higher cost of goods to ultimate purchasers. Several published studies have been directed to the expression of plant virus capsid proteins in a plant in an effort to confer resistance to viruses. See, e.g., Abel et al., Science 232:738-743 (1986); Cuozzo et al., Bio/Technology 6:549-557 (1988); Hemenway et al., EMBO J. 7:1273-1280 (1988); Stark et al., Bio/Technology 7:1257-1262 (1989); and Lawson et al., Bio/Technology 8:127-134 (1990). The transgenic plants exhibited resistance only to the homologous virus and related viruses, however, and not to unrelated viruses. Kawchuk et al., Mol. Plant-Microbe Interactions 3(5):301-307 (1990), disclose the expression of wild-type potato leafroll virus (PLRV) coat protein gene in potato plants. Although the infected plants exhibited resistance to PLRV, all of the transgenic plants that were inoculated with PLRV became infected with the virus and thus allowed for the continued transmission of the virus such that high levels of resistance could not be expected. See U.S. Pat. No. 5,304,730.
Pokeweed antiviral protein (PAP) is a 29-kDa Type I ribosome-inhibiting protein (RIP) found in the cell walls of Phytolacca americana (pokeweed). See, Wang et al., Adv. Virus Res. 55:325-356 (2000). It is a single polypeptide chain that catalytically removes a specific adenine residue from a highly conserved stem-loop structure (i.e., the α-sarcin loop) in the 28S rRNA of eukaryotic ribosomes, thus interfering with Elongation Factor-2 binding and blocking cellular protein synthesis. More specifically, PAP removes an adenine base by cleavage of the N-glycosidic bond at A4324 in rat 28 S rRNA and at homologous sites on ribosomes from other organisms. See, e.g. Irvin et al., Pharmac. Ther. 55:279-302 (1992); Endo et al., Biophys. Res. Comm. 150:1032-1036 (1988); and Hartley et al., FEBS Lett. 290:65-68 (1991). PAP recognizes and binds to the ribosomal protein L3 that is essential for subsequent depurination of the α-sarcin loop. See, Hudak et al., J. Biol. Chem. 274:3859-3864 (1999).
PAP protein confers resistance to a broad spectrum of viruses when expressed in crop plants, yeast and cultured human cells. Lodge et al., Proc. Natl. Acad. Sci. USA 90:7089-7093 (1993), report the Agrobacterium tumefaciens-mediated transformation of tobacco with a cDNA encoding wild-type pokeweed antiviral protein (PAP) and the resistance of the transgenic tobacco plants to unrelated viruses. Lodge also reports, however, that the PAP-expressing tobacco plants (i.e., above 10 ng/mg protein) tended to have a stunted, mottled phenotype, and that other transgenic tobacco plants that accumulated the highest levels of PAP were sterile. Since that time, Applicant has found that various PAP mutants provide comparable resistance to plant pests such as viruses and fungi but are less toxic than wild-type PAP. See, U.S. Pat. Nos. 5,756,322; 5,880,329 and 6,137,030. The PAP mutants disclosed in the prior art that exhibited less cytotoxicity (e.g., phytotoxicity) than wild-type PAP also exhibited the capability of depurinating the cell ribosomes. The belief was that cytotoxic effect was a result of translation inhibition due to depurinated rRNA.